Soutenance de thèse Adrien Lesage

The voltage-gated sodium channels NaV1.5 are responsible for the initiation and fast propagation of cardiac action potentials, and dysregulations of this channel underlie diverse forms of inherited or acquired cardiac disease. While phosphorylation of the NaV1.5 channel pore-forming subunit is extensive and recognized to mediate several molecular aspects of channel expression and function, the regulation of NaV1.5 channels by phosphorylation of its accessory proteins remains largely unexplored. A mass spectrometry-based phosphoproteomic analysis of mouse ventricular NaV1.5 channel complexes was undertaken to identify the native phosphorylation sites of NaV1.5 channel accessory proteins. Among the various accessory proteins identified in NaV1.5 channel complexes, the Fibroblast Growth Factor Homologous Factor 2 (FHF2) showed extensive phosphorylation at 9 specific sites. In order to determine the functional roles of these newly-identified FHF2 phosphorylation sites, we developed two novel cellular models in which the expression of endogenous FHF2 is knockdowned in isolated neonatal mouse ventricular cardiomyocytes using shRNA-expressing adenoviruses, or adult ventricular cardiomyocytes isolated from cardiac-specific FHF2 knockdown mice. The expression of FHF2 was then rescued in both cellular models using adenoviruses expressing the major ventricular FHF2 isoform, FHF2-VY, in its wild-type (WT), phosphosilent (mutations to alanine) or phosphomimetic (mutations to glutamate) forms at specific sites  Whole cell voltage-clamp analyses demonstrated that FHF2 knockdown accelerates the rate of closed-state and open-state inactivation of NaV channels in both neonatal and adult cardiomyocytes. Interestingly, FHF2 knockdown also shifted the voltage-dependence of NaV channel activation towards hyperpolarized potentials in neonatal cardiomyocytes. Although the rescue of FHF2 with WT FHF2-VY restored the inactivation properties of NaV channels in both neoanatal and adult cardiomyocytes, no restoration of the activation properties was obtained in neonatal cardiomyocytes, suggesting the involvement of another FHF2 isoform in regulating the activation properties of NaV channels in neonatal cardiomyocytes. However, similar to WT FHF2-VY, each of the analyzed FHF2-VY phosphomutants restored the inactivation properties of NaV channels in both cellular models, preventing to identify roles for FHF2 phosphorylation sites in regulating NaV1.5 channels. Further investigation also demonstrated that FHF2 knockdown increases the late Na+ current in adult cardiomyocytes, which was similarly restored with WT and phosphosilent FHF2-VY. Taken together, our results demonstrate that ventricular FHF2 is highly phosphorylated, implicate critical and differential roles for FHF2 in regulating NaV1.5 channels in neonatal and adult ventricular cardiomyocytes, and suggest that the regulation of NaV1.5 channels by FHF2 phosphorylation is certainly highly complex.